The 174-base bacteriophage phi29 prohead RNA (pRNA) is essential for in vitro packaging of the 19 kilobase pair DNA-gene product 3 complex (DNA-gp3) into the viral precursor capsid or prohead. pRNA forms a novel cyclic hexamer by intermolecular base pairing of identical molecules, and this hexamer binds to the head-tail connector of the prohead. pRNA is hypothesized to function in docking of the DNA-gp3 and the prohead, in recognition of the left end of DNA-gp3 to initiate packaging, and as a component of the DNA translocating ATPase. pRNA exits the DNA-filled head during neck and tail assembly, and it is not a part of the mature virion. Study of the structure and function of this RNA-dependent DNA packaging machine may have general significance for assembly of other viruses, including mammalian viruses. The ultimate goal of the research is to determine the mechanisms by which pRNA constitutes the phi29 DNA packaging machine and joins with the connector and packaging ATPase to catalyze DNA-gp3 translocation into the prohead. The aims of the current project are to: 1) study the gp16 interactive domain on pRNA by ribonuclease footprinting and in vitro selection of aptamers for gp16 binding; 2) investigate pRNA as the determinant of orientation of DNA packaging; 3) study the structure of pRNA hexamers and pRNA complexed to connectors or gp16 by transmission and scanning transmission electron microscopy; 4) use Fe(II)-EDTA as a probe of tertiary structure of free pRNA and pRNA engaged in DNA packaging; 5) refine the molecular models of pRNA; 6) produce diffraction quality crystals of pRNA and pRNA complexed to connectors or gp16; and 7) characterize the DNA packaging RNAs of phages lambda and T3.